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Lecture 7 and 8 PCR and Hybridization Based Markers

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Aksh Tiwari
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39 views

Lecture 7 and 8 PCR and Hybridization Based Markers

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Aksh Tiwari
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© © All Rights Reserved
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PCR and hybridization based

markers

Dr. Thamilarasi K
Senior Scientist
Agri-bioresources Augmentation Division
ICAR – National Institute of Secondary Agriculture
Namkum, Ranchi
Applications of markers
• Phylogenetic studies
• Trait identification and mapping
• DNA fingerprinting
• Genetic diagnosis
• Expression profile
• Varietal identification, strain identification
• Study of genome
• Gene mapping/Gene tagging
• Seed testing
• Identifying QTL (Quantitative Trait Loci)
• Marker assisted selection
• Molecular breeding
Molecular Markers

• A molecular marker usually is a fragment of DNA within a genome which can


be easily identified.
• Genetic alterations such as mutation, insertion and deletion can be
identified.
• They may or may not represent the target genes themselves but can act as
signs or flags for the genes.
• Markers that are located to close proximity of the genes that are tightly
linked to the genes are called tags.
• All molecular markers occupy specific genomic positions within
chromosomes (like genes) called loci (singular locus).
• They are consistent and not affected by environmental factors.
Features of an ideal molecular marker

• Polymorphic: A polymorphism (more than one form) is a detectable and


heritable variation at a locus. It arises as a result of several classes of
mutations like substitution (as little as a single nucleotide), genomic
rearrangements due to insertion or deletion or errors in replication of arrays
of tandemly-repeated DNA.
• Reproducible: should give similar results at different time and place.
• Preferably displays co-dominant inheritance
• Frequent occurrence in the genome
• Closely linked to the trait of interest
• Easy access and detection: Should demonstrate measurable differences in
expression between trait types and/or alleles of interest, early in the
development of the organism.
Co-dominant marker Dominant marker

Codominant markers can clearly discriminate between


homozygotes (AA ) and heterozygotes (Aa) whereas
dominant markers do not.
PCR based markers

• RAPD – Random Amplified Polymorphic DNA


• SCAR – Sequence Characterized Amplified Region
• ISSR – Inter Simple Sequence Repeats
• SSR – Simple Sequence Repeats (also called Microsatellite markers)
• CAP- Cleaved Amplified Polymorphic Sequences
• SNP – Single Nucleotide Polymorphism
• EST – Expressed Sequence Tags
• STS – Sequence Tagged Site
• DArT - Diversity Arrays Technology (DArT)
Hybridization based marker

• RFLP – Restricted Fragment Length Polymorphism

PCR and hybridization based marker


• AFLP – Amplified Fragment Length Polymorphism
RAPD – Random Amplified Polymorphic DNA

• RAPD detects nucleotide sequence polymorphisms in DNA by using a single


primer of arbitrary nucleotide sequence (random).
• A single primer anneals to the genomic DNA at two different sites on
complementary strands of DNA template and if the sites are within
amplifiable range, a discrete DNA product is formed during PCR.
• Primers used are generally 10 bp random sequences.
• Amplified fragments are usually within 0.5 – 5 Kb size range.
• Variations are primarily due to variations in the primer annealing sites,
sometimes due to difference in the length of the amplified sequences
between primer annealing sites.
• They are dominant markers.
SCAR – Sequence Characterized Amplified Region

• In this technique, the RAPD marker is sequenced,


and longer primers are designed (22-24 nucleotides
long) for specific amplification of particular locus.
• Since longer primers are used, it is reproducible
unlike that of RAPDs.
• Informative marker and exhibits co-dominance and
at times dominance character.
• Limitations: Needs sequencing and takes time to
develop
• Relatively few in number.
SSR- Simple Sequence Repeats
• SSRs or microsatellites are sections of DNA consisting of tandem repeats of mono, di, tri,
tetra, penta and hexa nucleotides.
• They are hyper variable, abundant and arranged throughout the genome of the
eukaryotes.
• If nucleotide sequences of the flanking regions of the repeats are known, specific
primers (generally 20-25 bp long) can be designed to amplify the SSRs by PCR.
• Polymerase slippage during DNA replication or slipped strand misrepairing is
considered to be the main cause of variation in the number of repeat units of SSRs,
resulting in length polymorphism that can be detected by PCR.
• They are codominant, locus specific markers, highly polymorphic, multi allelic and they
are amenable for multiplexing.
• Primers of closely related species can be used.
• Primers development is cost and labour intensive.
Regions for SSR primer development

Representative image of SSR markers


CAPs – Cleaved Amplified Polymorphic Sequences

• CAPs are DNA fragments amplified by PCR using specific 20-25 bp primers, followed
by digestion of the PCR products with a restriction enzyme.
• PCR primers can be synthesized based on the sequence information available in
genomic or cDNA databanks or cloned RAPD bands.
• CAPs are also referred as PCR-RFLP (PCR-Restriction Fragment Length Polymorphism)
• Co-dominance and highly reproducible
• No need to use radioactive material as in RFLP analysis
• Limitation: CAPs are more difficult to find because of the limited size of the amplified
fragments and sequence data is needed for synthesis of the primers.
• Application: Predominantly in gene mapping studies.
SNP – Single Nucleotide Polymorphism
• SNPs (pronounced “snips”) are DNA sequence variations that occur when a single nucleotide
(A,T,C, or G) in the genome is altered.
• E.g. 5’ AAGGTACA 3`
5’ AAGGACA 3`
• For a variation to be considered as SNP, it must occur in at least 1% of the population
• Type of mutation: Transition – Conversion of purine to purine (A G) and pyrimidine to
pyrimidine (C T) Transversion: Purine for pyrimidine or pyrimidine for purine
• Types of SNPs: Regulatory SNPs (rSNPs)– SNPs in regulatory regions (promoter SNPs, intron
SNPs).
• SNPs in genic regions – Anonymous SNPs (Functional effect is not known), Candidate SNPs
(functional effect is known)
• Sequence information is required to design allele specific primers. 1. Library construction and
sequencing or 2. through the screening of readily available sequence databases.
• Advantage is the possibility of high throughput automation through multiplexing
• Useful for cultivar discrimination in crops where it is difficult to find polymorphism such as in
cultivated tomato and saturate linkage maps (E.g. SNPs were used to construct high density
linkage maps for easy to score DNA markers in Arabidopsis thaliana)
RFLP

RFLP – Hybridization based marker


R-Restriction
F-Fragment
L-Length
P-Polymorphism
Restriction enzyme
Procedure

• Step 1: Collection of sample


• Step 2: DNA extraction
• Step 3 : Restriction digestion
• Step 4: Gel electrophoresis (Southern blot)
• Step 5: Denaturation (NaoH)
• Step 6: Blotting
• Step 7: Blocking
• Step 8: Hybridization
• Step 7: Visualization
Application
Co-dominant: distinguish the homozygous and heterozygous DNA
Sequence specific: Probe are sequence specific
Genomic abundance: restriction sites are abundant throughout the genome
Reference

• Samuel Amiteye, Basic concepts and methodologies of DNA marker


systems in plant molecular breeding, Heliyon, Volume 7, Issue 10,
2021, e08093, ISSN 2405-8440,
https://doi.org/10.1016/j.heliyon.2021.e08093.

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